Method for preparing chicken immortalized cell line

Abstract

The invention provides a method for preparing a chicken immortalized cell line. The method comprises the following steps: constructing a plasmid system of conditional over-expression TERT (Telomerase Reverse Transcriptase), carrying out ovarian injection and breeding to obtain a transgenic chicken of the conditional over-expression TERT, and carrying out separation and induction to obtain the chicken immortalized cell line. The obtained chicken immortalized cell line has the characteristics of primary cells and is susceptible to viruses; the method can be used for virus reproduction and detection, influenza virus vaccine production and the like, can obtain immortalized cell lines of all organs and all parts of the chicken in a short time, and has a good application prospect in the vaccine industry.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to a method for preparing a chicken immortalized cell line. Background Art

[0002] Normal animal cells are in a dynamic balance of proliferation, differentiation, aging and apoptosis. After a limited number of passages under in vitro culture conditions, cells derived from normal tissues will stop proliferating and age and die. This is the natural life span of cells, known as the Hayflick limit (Hayflick, 1965). Cell immortalization refers to the process in which cells cultured in vitro escape from normal proliferation and aging crises spontaneously or under the influence of external factors, thereby acquiring unlimited division and proliferation capabilities.

[0003] The study of cell immortalization is not only conducive to understanding the molecular mechanism of cell proliferation and aging, but also lays a solid foundation for exploring tumor treatment and organ transplantation. Thanks to the characteristics of immortalized cells that can be propagated indefinitely, various methods can be used to immortalize cells that are difficult to propagate, proliferate slowly, and are prone to aging, providing more cell resources and good in vitro cell models for various research fields.

[0004] The main mechanisms of cell immortalization include: activation of oncogenes or inactivation of tumor suppressor genes caused by physical and chemical factors, stable activation of telomerase, and tumorigenic viruses such as SV40 virus, Epstein-Barr virus, and human papillomavirus HPV. Accordingly, based on the above mechanisms, exogenous immortalization genes can be artificially introduced into target cells through transfection technology, tumor suppressor genes can be knocked out, and immortalization-related gene mutations can be induced to establish stable immortalized cell lines.

[0005] Immortalized chicken cells can be used for virus propagation, detection, and subsequent influenza virus vaccine production. At present, some spontaneously immortalized cell lines, such as DF-1, cannot be used for the preparation of influenza virus vaccines because their growth rate varies over time. In addition, cells susceptible to different viruses are different, and there are currently too few immortalized cell types that can be used to prepare influenza virus vaccines. Therefore, it is urgent to establish a variety of chicken immortalized cell lines that are susceptible to viruses and have primary cell characteristics. Summary of the invention

[0006] The present invention provides a method for preparing a chicken immortalized cell line, which is used to solve the defects of the prior art that the chicken immortalized cells are few in variety and difficult to obtain, and realizes efficient and rapid acquisition of the chicken immortalized cell line.

[0007] In a first aspect, the present invention provides a method for preparing a chicken immortalized cell line, comprising injecting a plasmid that conditionally overexpresses TERT (Telomerase Reverse Transcriptase) into the ovaries of unfertilized chickens, artificially inseminating the chickens injected with the conditional overexpression TERT plasmid, collecting eggs and hatching them into chicks, screening to obtain transgenic chicks containing conditional overexpression TERT sequences, and inducing transgenic chick cells to obtain a chicken immortalized cell line.

[0008] Preferably, the above method comprises the steps of: (1) Construction of a plasmid system for conditional overexpression of TERT; (2) Obtain transgenic chickens that conditionally overexpress TERT; (3) Immortalized chicken cell lines were obtained by isolating transgenic chicken cells that induced conditional overexpression of TERT.

[0009] Preferably, in step (1) of the above method, the vector used to construct the plasmid system for conditional overexpression of TERT is a piggyBac vector, and the promoter used is a CAG promoter.

[0010] PiggyBac transposon can mediate efficient integration of exogenous genes into the genome with the help of PB transposase, effectively reduce transgenic silencing and avoid safety risks caused by the use of viral vectors. PiggyBac has a high gene insertion efficiency and can be stably integrated into the host genome, especially suitable for long-term expression. CAG promoter is an artificially constructed combination promoter. It is a combination of the enhancer of cytomegalovirus CMV, chicken β-actin promoter and intron. CAG promoter is suitable for a variety of cell types and tissues, so it can achieve stable gene expression in different developmental stages or tissues of chickens. Since the components of CAG promoter are selected from the human and chicken genomes, it can effectively drive expression between different species (including chickens).

[0011] Further preferably, the plasmid system for conditional overexpression of TERT also contains a loxP element and a nucleotide fragment encoding the TERT protein, the coding order of all sequences in the loxP element is opposite to the coding direction of the TERT protein initiated by the CAG promoter, and the loxP element is located between the CAG promoter and the nucleotide fragment encoding the TERT protein.

[0012] The loxP element consists of two inverted repeat sequences with 13 base pairs and a spacer sequence with 8 base pairs, which can be recognized by Cre recombinase. When the two inverted repeat sequences in the loxP element are in the same direction, Cre recombinase will delete the sequence between the loxP elements. The coding direction of the loxP element in the present application is opposite to the CAG promoter and the nucleotide fragment encoding the TERT protein and is located between the two. When the Cre recombinase is not added, the transcription of the TERT gene is blocked. After the Cre recombinase is added to delete the sequence between the loxP elements, the TERT gene can be transcribed and translated normally.

[0013] In an embodiment of the present application, the loxP element also includes an expression system of the G418 resistance gene Neo, including a PGK promoter, a Neo gene and a polyA sequence, which is used to confirm whether the constructed conditional overexpression TERT plasmid is successfully transfected, and the transcription termination function of the polyA signal between the loxP elements blocks the activation of downstream genes by the promoter.

[0014] In some embodiments of the present application, the plasmid also contains a GFP sequence. When the Cre recombinase deletes the sequence between the loxP elements, it will cause the expression of the GFP green fluorescent protein to change, which is used to confirm whether the sequence between the loxP elements can be deleted by the Cre recombinase.

[0015] In some embodiments of the present application, the above-mentioned GFP sequence is located outside the loxP element and is connected to the TERT sequence through an IRES sequence for co-expression, so that when the loxP element is present, neither GFP nor TERT is expressed. When the Cre recombinase is added, the sequence between the loxP elements is deleted, and both GFP and TERT are expressed normally.

[0016] Preferably, obtaining a transgenic chicken that conditionally overexpresses TERT in step (2) of the above method comprises the following steps: (a) injecting the conditional overexpression plasmid of TERT into the ovaries of unfertilized hens; (b) artificially inseminating the hen injected with the plasmid for conditional overexpression of TERT; (c) The artificially inseminated eggs were collected and incubated to screen for transgenic chickens that conditionally overexpressed TERT.

[0017] Further preferably, before the injection in step (a) of the above method, the conditional overexpression plasmid of TERT (including the plasmid carrying TERT and the PB transposase plasmid) is mixed with the liposome, and the injection target is the small white follicles in the chicken ovary or the small yellow follicles containing a small amount of yolk. The liposome used in the embodiment of the present application is lipo2000, and the mixing ratio is 20 μg of plasmid and 40 μL of Lipo2000.

[0018] Preferably, the method for screening and obtaining transgenic chickens that conditionally overexpress TERT in step (c) of the above method is to extract the comb genome of the chicks hatched from the breeding eggs and perform PCR detection.

[0019] The pair of primer sequences used in the PCR detection are shown in SEQ ID NO.6 and SEQ ID NO.7; and / or The PCR detection system is as follows: DNA template 1 μl, Primer Mix 0.75 μl, dNTP 0.15 μl, GoTaq 0.15 μl, MgCl 2 1μL, Buffer 2.5μL, Water 6.95μL; and / or The PCR detection program is: preheating at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, further extension at 72°C for 7 min, and holding at 4°C.

[0020] In the examples of the present application, probes for the GFP gene and the TERT gene were used to perform Southern Blot hybridization experiments on the comb genomes of chickens that were PCR positive for GFP and TERT genes in the offspring after ovarian injection to verify the presence of the transgene.

[0021] Preferably, in step (3) of the above method, the method for isolating and inducing transgenic chickens that conditionally overexpress TERT to obtain a chicken immortalized cell line is to isolate cells from a desired part of the transgenic chickens that conditionally overexpress TERT and add Cre recombinase for induction.

[0022] In a second aspect, the present invention provides a chicken immortalized cell line, which is prepared by the above method for preparing a chicken immortalized cell line.

[0023] In a third aspect, the present invention provides the use of the above method or the above chicken immortalized cell line in any of the following: (1) High-throughput screening of drugs and evaluation of drug toxicity and efficacy; (2) Biopharmaceuticals, where the drug is a vaccine or an antibody; (3) Cell fermentation; (4) Reproduction of viruses; (5) Construct disease models; (6) Production of replacement tissues or organs; (7) Production of artificial meat The beneficial effects of the present invention are as follows: The method for preparing a chicken immortalized cell line provided by the present invention can solve the defects of the prior art that chicken immortalized cells are few in variety and difficult to obtain, achieve efficient and rapid acquisition of chicken immortalized cell lines, and can obtain various chicken immortalized cell lines in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of three plasmids constructed in Example 1 of the present invention.

[0026] Figure 2 It is a diagram of the inverted fluorescence observation results of chicken DF-1 cells transfected with pJJH11 plasmid before and after Cre recombinase induction in Example 2 of the present invention.

[0027] Figure 3 It is a diagram of the inverted fluorescence observation results of chicken DF-1 cells transfected with pJJH14 plasmid before and after Cre recombinase induction in Example 2 of the present invention.

[0028] Figure 4 It is the RT-PCR result diagram of three experimental groups in Example 2 of the present invention.

[0029] Figure 5 This is a graph showing the electrophoresis results of the products of PCR amplification of the genomic DNA of the comb of a transgenic chicken that conditionally overexpresses TERT obtained in Example 3 of the present invention.

[0030] Figure 6 Schematic diagram of the PJJH11 plasmid and primer structure used in Example 4 of the present invention.

[0031] Figure 7 This is a diagram showing the Southern Blot results of TERT-positive chicks in Example 4 of the present invention.

[0032] Figure 8 This is a diagram showing the results of the transfer of exogenous TERT and GFP sequences into various organs of TERT-positive chicks in Example 5 of the present invention.

[0033] Fig. 9 This is the appearance picture of the TERT positive chick in Example 5 of the present invention.

[0034] Fig.10This is a result diagram of the cell appearance and DFP protein expression of primary kidney cells extracted from TERT-positive chickens in Example 5 of the present invention before and after Cre enzyme induction. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Example 1 Construction of a plasmid system for conditional overexpression of TERT A series of chicken TERT conditional overexpression vectors of piggyBac transposon system were constructed. The vector diagram is shown in the figure Figure 1 As shown, the pJJH10 plasmid (SEQ ID NO.1) does not contain the GFP fluorescent reporter element, and the expression of the chicken TERT gene can be restored by removing the G418 drug-resistant gene Neo between the CAG promoter and the loxP site between the chicken TERT gene sequence using Cre recombinase.

[0037] The chicken TERT gene and the fluorescent reporter gene GFP in the pJJH11 plasmid (SEQ ID NO.2) are conditionally expressed in a Cre recombinase-mediated form. In the absence of Cre recombinase, neither the chicken TERT gene nor the green fluorescence is expressed.

[0038] In the pJJH14 plasmid (SEQ ID NO.3), the chicken TERT gene is conditionally expressed mediated by Cre recombinase. In the absence of Cre recombinase, GFP is expressed, but the chicken TERT gene is not expressed.

[0039] The PB transposase expression vector pPBase (Xu C, Qi X, Du X, et al. piggyBac mediates efficient in vivo CRISPR library screening for tumorigenesis in mice[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(4):201615735.) can mediate the efficient and stable integration of the gene sequence between the two LTRs in the above plasmid into the genome.

[0040] Example 2 Plasmid transfection for conditional overexpression of TERT and Cre induction effect test The above-mentioned pJJH10, pJJH11, and pJJH14 plasmids were combined with the PB transposase expression vector pPBbase to be transfected into chicken DF-1 cells as follows: The plasmids in each group were as follows: experimental group 1 (pJJH10 3μg, pPBase 2μg), experimental group 2 (pJJH11 3μg, pPBase 2μg), and experimental group 3 (pJJH14 3μg, pPBase-4PB 2μg).

[0041] Electroporation: The electroporator was 2B-Nucleofector Device (Lonza) and the program was T-023.

[0042] Drug screening: 16 h after electroporation, add 700 μg / mL G418 for screening for 3 days.

[0043] Observation of the results of electroporation and Cre recombinase induction characterized by GFP: After the drug screening, the cell pellet was collected, and the cells of experimental group 2 and experimental group 3 with the GFP sequence in the plasmid were taken. According to the instructions of TAT-Cre (Merck SCR508), 2×10 cells were plated in 12-well plates and counted in each well. 5 Then, 2 μmol (i.e., 1 μmol / ml) Cre recombinase was added to each well. After 48 hours, the expression of GFP before and after adding the recombinase was photographed under an inverted fluorescence microscope. The results of experimental group 2 are as follows: Figure 2 The results of experimental group 3 are shown in Figure 3 shown.

[0044] The specific operation of fluorescent quantitative PCR for chicken TERT is as follows: Total RNA of chicken DF-1 cells cultured for 48 h after transfection was extracted using a cell RNA extraction kit (Qiagen RNeasy mini kit) according to the instructions. Fluorescent quantitative RT-PCR was performed on the chicken TERT gene using an RNA reverse transcription kit (Qiagen QuantiTect ReverseTranscription Kit) with chicken Actin as the internal reference gene. The sequences of fluorescent quantitative RT-PCR primers PJ68-RT-5F and PJ68-RT-5R are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0045] PJ68-RT-5F: 5'-CTGAGGCTGGCTAAGGGAAACCACA-3' (SEQ ID NO. 4).

[0046] PJ70-RT-5R: 5'-TTAGCGAAGTGGTCCCTGATTCCGA-3' (SEQ ID NO. 5).

[0047] The fluorescence quantitative RT-PCR reaction system is shown in Table 1: Table 1 The reaction system was prepared on ice, and three technical replicates were performed for each sample.

[0048] The fluorescence quantitative RT-PCR program is shown in Table 2: Table 2 The results of fluorescence quantitative RT-PCR were as follows Figure 4 shown.

[0049] Depend on Figure 2 It can be seen that some cells showed green fluorescence, proving that after the addition of Cre, loxP in experimental group 2 was deleted, thereby allowing the expression of TERT and GFP elements.

[0050] Depend on Figure 3 It can be seen that the proportion of green fluorescence has decreased, but due to the limited efficiency of TAT-Cre, negative cells are still proliferating. Figure 3 More fluorescence can also be seen, proving that after the addition of Cre, loxP in experimental group 3 was deleted, thereby deleting the GFP element therein.

[0051] Depend on Figure 4 It can be seen that after adding Cre, the elements between loxP were deleted, which significantly increased the expression of TERT in the three experimental groups. Since the determination of TERT transfer in experimental groups 1 and 3 was not as convenient as that in group 2, the pJJH11 plasmid of experimental group 2 was finally selected.

[0052] Example 3 Preparation of transgenic chickens with conditional overexpression of TERT by chicken ovary injection Fifteen Loch Island White hens around 18 weeks old and close to laying were selected. The hens were injected with 0.1 mL of ketamine for sedation, and then intubated with isoflurane for respiratory anesthesia. The incision was located between the second and third ribs on the left side of the chicken from back to front, and the ovaries could be observed after incision. The oocytes in the hen's ovaries were in the stage of small white follicles or small yellow follicles with a small amount of yolk, and large yellow follicles full of yolk that were difficult to inject, and small white follicles or small yellow follicles with a small amount of yolk.

[0053] The pJJH11 plasmid and liposome (pJJH11 20μg, Lipo2000 40μL) were co-injected into the ovaries of 15 18-week-old Lodow White hens. Wild-type rooster semen was collected for artificial insemination of the hens that recovered after injection, and the breeding eggs were collected for incubation.

[0054] Using specific PCR primers for chicken TERT and GFP, PCR detection was performed on the comb genome of the offspring of ovarian injected hens. The specific primers for chicken TERT were marked as PJ373 and PJ374, the primer sequences were shown in SEQ ID NO.1 and SEQ ID NO.2, and the amplified product size was 641 bp; the specific primers for GFP were marked as WS211 and WS212, the primer sequences were shown in SEQ ID NO.5 and SEQ ID NO.6, and the amplified product size was 354 bp. The positive control of the PCR experiment was the injection plasmid pJJH11.

[0055] PJ373: 5'-TTGCTCACCATGGTTGTGGC-3' (SEQ ID NO. 6).

[0056] PJ374: 5'-TGTGCCAGGACTTTAAGACC-3' (SEQ ID NO. 7).

[0057] WS211: 5'-AAGCTGACCCTGAAGTTCATCTGC-3' (SEQ ID NO. 8).

[0058] WS212: 5'-CTTCTGCTTGTCGGCCATGATATAG-3' (SEQ ID NO. 9).

[0059] The PCR reaction system is shown in Table 3: The PCR program is shown in Table 4: After the PCR amplification product was purified, nucleic acid electrophoresis was performed. Figure 5 As shown, both GFP and TERT sequences can be detected with the correct size, indicating that the exogenous plasmid pJJH11 was successfully integrated into the chicken somatic cells.

[0060] Example 4 Southern Blot Verification of Transgenic Chickens Conditionally Overexpressing TERT The TERT and GFP primer systems in Example 3 were used, and the pJJH11 plasmid was used as a template. The probes were synthesized using the PCRDIG probe synthesis kit (Roche 11636090910) according to the instructions. The two probes were used to perform Southern Blot hybridization experiments on the comb genomes of chicks with GFP and TERT sequence PCR positive in the offspring after ovarian injection to verify the presence of the transgene.

[0061] The plasmid control (1 μg) and chicken blood genome (15 μg) were digested with restriction endonuclease SpeI. The plasmid used as the control was the ovarian injection plasmid pJJH11. The plasmid and primer structures were as follows: Figure 6 As shown. The exogenous transgenic plasmid fragment obtained by hybridization using TERT and GFP amplification products as probes is about 9.7 kb after SpeI digestion. Since the plasmid is inserted into the genome, the SpeI digestion site in the PGK promoter will be digested, and the other digestion site is at a random position on the genome, so the final fragment size should be greater than 9.7 kb.

[0062] The specific steps of Southern Blot are as follows: 1. Digest the genome with 15 μg of each sample.

[0063] 2. Electrophoresis: Use 0.8% agarose gel, run at 70V out of the gel well, and then run electrophoresis at 22V for about 11 hours. The positive control is plasmid, and the loading amount is 100pg.

[0064] 3. Add denaturation solution and denature for 1 hour at a speed not exceeding 50 rpm.

[0065] 4. Add transfer buffer and wash the gel for 15 minutes.

[0066] 5. Transfer: The nylon membrane should be rinsed with transfer solution in advance, and the sponge should be washed with water first, and then washed with transfer solution and soaked. Try to keep it flat with the sponge. The order from bottom to top is: towel - filter paper - membrane - glue - wrap with plastic wrap around - filter paper - sponge.

[0067] 6. Neutralize the nylon membrane for 10 minutes and gently put it in. At this time, the DNA is not tightly bound to the nylon membrane.

[0068] 7. UV crosslinking for 3-5 minutes, with the front side of the membrane facing up, 2000 energy, continuous irradiation for 3-5 minutes, and drying for 15 minutes.

[0069] 8. Pre-hybridization: Wear gloves and place the membrane into the hybridization tube (front of the membrane facing inwards), add 15mL DIG hybridization solution (powder, add 65ml ddH 2 O, absorbs heat when dissolved), and hybridize in a 45℃ hybridization tube for 1h.

[0070] 9. Denature probe: Take 20 μl (60 ng / μl) DIG-labeled probe and add 50 μl ddH 2 O 100℃ for 10 min, immediately put on ice and keep in ice bath for more than 10 min.

[0071] 10. Hybridization: Preheat 10 mL of hybridization solution at 45°C, add all the denatured 70 μl probe, mix well, pour out the pre-hybridization solution in the hybridization tube, add new 10 mL of hybridization solution, and hybridize at 45°C for 17-20 hours.

[0072] 11. Wash the membrane: Pour out the hybridization solution and fill it with about 100 ml of 2×SSC, 0.1% SDS solution preheated at 50℃. Wash in a hybridization oven at 50℃ for 15 minutes. Repeat once.

[0073] 12. Pour out the original solution and fill it with about 100 ml of 0.5× SSC, 0.1% SDS preheated at 68°C. Wash in a hybridization oven at 68°C for 30 min. Repeat once.

[0074] 13. Wash the membrane: Pour out the original solution, add 1× washing buffer, and wash the membrane for 3-5 min.

[0075] 14. Blocking: Pour out the original solution and add 50mL 1× blocking solution to block for 1h.

[0076] 15. Hybridization: Pour out the original solution, carefully add 50 mL of antibody solution, and hybridize for 30 minutes.

[0077] (Each hybridization membrane needs to be prepared at a dilution of 1:50,000, add 1 μl of antibody to the remaining 50 mL of blocking solution in the second step, and mix in a 50 ml centrifuge tube).

[0078] 16. Wash the membrane: Pour out the original solution and add 100 mL of Washing buffer for 15 min. Repeat once.

[0079] 17. Pour off the original solution and add 100 mL of Detection buffer for 2-5 min.

[0080] 18. Take out the membrane and place it in a clean plastic bag. Evenly add a thin layer of CDP-STAR on the membrane to remove bubbles and excess CDP-STAR. Seal the membrane and incubate at 37°C for 10 minutes (do this in an incubator and then store it in a new hybridization bag).

[0081] 19. Membrane scanning: Membrane scanning time 10 s-3 min.

[0082] The results are as follows Figure 7 As shown, the 8 PCR-positive offspring chicks could all be hybridized with two different probes to obtain positive bands, and the fragment size was consistent with expectations, which could be confirmed to be exogenous GFP and chicken TERT transgenic positive offspring chicks.

[0083] Example 5 Verification of TERT sequences in various tissues and cells of transgenic chickens that conditionally overexpress TERT The chickens with positive TERT sequence PCR in the comb genome of Example 3 ( Fig. 9 ) Dissect and obtain tissue samples from various organs, extract genomic DNA, and perform PCR detection on the genomic DNA of various organs using the primers and PCR program in Example 2. After the detection results are purified, nucleic acid electrophoresis is performed. The results are as follows Figure 8 shown.

[0084] Transgenic chicken primary kidney cells that conditionally overexpress TERT were isolated by trypsin digestion. According to the instructions of TAT-Cre (Merck SCR508), 2×10 cells were plated in 12-well plates and counted per well. 5 Then, 2 μmol (i.e., 1 μmol / ml) of Cre recombinase was added to each well and the expression of the chicken TERT gene was induced after 48 hours. Before and after the addition of Cre recombinase, the induction of the expression of the exogenous GFP gene in the chicken primary kidney cells was observed using an inverted fluorescence microscope. Fig.10 shown.

[0085] Depend on Figure 8 It can be seen that all organs of chickens with positive PCR results for TERT sequence in the comb genome carry exogenous conditionally overexpressed TERT sequences, and are capable of being induced into immortalized cell lines by Cre recombinase.

[0086] Depend on Fig.10 It can be seen that the structural morphology of primary renal cells changed after Cre recombinase induction. In the inverted fluorescence photography, the cells induced by Cre recombinase had obvious green fluorescence, indicating that the sequence between the loxP elements was successfully deleted and the TERT and GFP sequences could be expressed smoothly.

[0087] The immortalized cell line obtained in the present application has unlimited proliferation ability, can achieve long-term culture, and can still maintain its original characteristics after multiple passages. It has a stable genotype and retains the characteristics of primary cells in terms of cell morphology, division time and cell surface antigens.

[0088] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a chicken immortalized cell line, characterized in that: The method includes injecting a plasmid that conditionally overexpresses TERT into the ovaries of unfertilized chickens, artificially inseminating the chickens injected with the conditional overexpression TERT plasmid, collecting eggs and hatching them into chicks, screening transgenic chicks containing conditional overexpression of TERT sequences, and inducing transgenic chick cells to obtain chicken immortalized cell lines.

2. The method according to claim 1, characterized in that Includes steps: (1) Construction of a plasmid system for conditional overexpression of TERT; (2) Obtain transgenic chickens that conditionally overexpress TERT; (3) Immortalized chicken cell lines were obtained by isolating transgenic chicken cells that induced conditional overexpression of TERT.

3. The method according to claim 2, characterized in that The vector used in constructing the plasmid system for conditional overexpression of TERT in step (1) is a piggyBac vector, and the promoter used is a CAG promoter.

4. The method according to any one of claims 1 to 3, characterized in that: The plasmid system for conditional overexpression of TERT contains a loxP element and a nucleotide fragment encoding the TERT protein. The coding order of all sequences in the loxP element is opposite to the coding direction of the TERT protein initiated by the CAG promoter. The loxP element is located between the CAG promoter and the nucleotide fragment encoding the TERT protein.

5. The method according to claim 2, characterized in that: The step (2) of obtaining transgenic chickens that conditionally overexpress TERT comprises the following steps: (a) injecting the conditional overexpression plasmid of TERT into the ovaries of unfertilized hens; (b) artificially inseminating the hen injected with the plasmid for conditional overexpression of TERT; (c) The artificially inseminated eggs were collected and incubated to screen for transgenic chickens that conditionally overexpressed TERT.

6. The method according to claim 5, characterized in that In step (a), the conditionally overexpressing TERT plasmid is mixed with the liposome before injection.

7. The method according to claim 5, characterized in that The method for screening and obtaining transgenic chickens that conditionally overexpress TERT in step (c) is to extract the comb genome of the chicks hatched from the breeding eggs and perform PCR detection; The pair of primer sequences used in the PCR detection are shown in SEQ ID NO.6 and SEQ ID NO.7; and / or The PCR detection system is: DNA template 1 μl, Primer Mix 0.75 μl, dNTP 0.15 μl, GoTaq 0.15 μl, MgCl2 1 μl, Buffer 2.5 μl, water 6.95 μl; and / or The PCR detection program is: preheating at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 30 cycles, further extension at 72°C for 7 min, and holding at 4°C.

8. The method according to claim 2, characterized in that: The method for isolating the transgenic chickens that conditionally overexpress TERT to obtain the chicken immortalized cell line in step (3) is to isolate cells from the desired part of the transgenic chickens that conditionally overexpress TERT and add Cre recombinase for induction.

9. A chicken immortalized cell line, characterized in that: The cell line is prepared by the method for preparing a chicken immortalized cell line according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 or the chicken immortalized cell line according to claim 9 in any one of the following: (1) High-throughput screening of drugs and evaluation of drug toxicity and efficacy; (2) Biopharmaceuticals, where the drug is a vaccine or an antibody; (3) Cell fermentation; (4) Reproduction of viruses; (5) Construct disease models; (6) Production of replacement tissues or organs; (7) Production of artificial meat.

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